JPH0767629A - Target gene substitution method - Google Patents

Abstract

PURPOSE:To enable sure and efficient substitution of a gene having desired structure for an arbitrary gene of an ES cell genom DNA and to get a transgenic animal useful in wide research and industrial fields. CONSTITUTION:A target gene region of an embryo stem cell genom DNA is substituted with a gene having a desired structure by using an embryo stem cell containing a target gene region of genom DNA homologously recombined with a DNA sequence containing one or more kinds of foreign gene exhibiting resistance and sensitivity to each of two different kinds of antibiotic substances, introducing a DNA fragment integrated with a gene having a desired structure into the embryo stem cell and homologously substituting the introduced DNA fragment for the homologous recombination sequence region of the embryo stem cell DNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an embryonic stem cell, a target gene replacement method using the embryonic stem cell, a substitute embryonic stem cell obtained by the method, and a transgenic animal. More specifically, the present invention relates to a target gene replacement method for selectively replacing an arbitrary gene of embryonic stem cell genomic DNA with a gene having a desired structure, and an animal individual in which a specific gene is replaced with a gene having a desired structure. It is a thing.

The target gene replacement method of the present invention can be applied not only as a research strategy in basic research fields such as genetics and embryology, but also as a new gene therapy. Further, the transgenic animal is useful as a disease model animal for elucidating human genetic diseases and developing therapeutic methods and therapeutic agents therefor.

[0003]

2. Description of the Related Art In recent years, there have been remarkable advances in gene manipulation techniques such as recombinant DNA, and their application to not only general biology but also medicine, engineering, agriculture, etc. is rapidly expanding. . In addition, regarding the gene itself, a great deal of knowledge has been accumulated from the analysis of its nucleotide sequence structure, and the research target nowadays extends to a huge amount of human genomic DNA which is said to be 3 billion base pairs.

However, in order to truly understand human biological phenomena by the various genes thus determined in structure, functional analysis of those genes at an individual level is indispensable. On the other hand, as many human diseases such as cancer are revealed to be caused by some abnormality in gene expression, laboratory animals used for medical research are also required to carry out gene analysis and gene manipulation. .

[0005] For these reasons, a technique capable of systematically expressing a specific gene (for example, a human unknown function gene or a disease-causing gene) in an animal individual and research on such an animal itself have been proposed. It is being actively done. An important achievement of such research is the so-called gene transfer method. This is, for example, a method in which a foreign gene DNA is physically introduced into a fertilized egg of a mouse, cultured to some extent in vitro, and then returned to the uterus of a female mouse to develop into an individual. It has become possible to obtain an animal (transgenic animal) in which a foreign gene is stably integrated in a part of the chromosome.

However, in the case of this method, it is impossible to integrate the transgene into a desired position of the animal chromosome, so that the transgene can be efficiently expressed at the individual level or the specificity of its expression function. Was difficult to identify. On the other hand, recently, a new gene transfer method has been developed in which any gene of an animal can be replaced with a desired structure by the transgene. That is the target gene transfer method (gene targeting: gene targe).
ting).

FIG. 8 is a process drawing of a target gene-introduced mouse using this method. In this target gene transfer method, as shown in FIG. 8, a foreign gene DNA is added to a mammalian (mouse) embryonic stem cell (Embryonic Stem Cell:
It may be referred to as an ES cell). The mouse ES cells are cells derived from the inner cell mass of the early embryo that eventually develops into a mouse individual, and can be cultured and proliferated outside the body in an undifferentiated state. Therefore, the foreign gene DNA was introduced into the ES cells in culture using electroporation or the like,
If these cells are injected into a blastocyst obtained from another strain of mouse and transplanted into a foster mother mouse, they will become part of the injected normal embryo and develop into an individual, resulting in a chimeric mouse (striped pattern). . If ES cell-derived cells have differentiated into the germ cells of this chimeric mouse, the properties of the ES cells are transmitted to the offspring, and a transgenic mouse in which any gene on its chromosome is replaced by a foreign gene (target gene transfer) Mouse) is obtained.

However, even with this method, ES
In most cases, foreign gene DNA introduced into cells is inserted at a random position on the chromosome, and the probability of recombination with the homologous gene (target gene) (homologous recombination) is about 1/1000 ( It is estimated to be about 10 ^-3 ). Therefore, in order to obtain an animal individual having a mutation derived from a foreign gene in a target gene, at the ES cell level,
It is necessary to select only homologous recombinants, and as a method therefor, the following selection methods using drug resistance have been proposed.

That is, in this method, the structure of the transgene is devised so that the gene expression regulatory region (promoter / enhancer) and polyA signal are not included in the sequence, and further, the drug G418 is treated. A DNA fragment is constructed by inserting the Neo resistance gene (neo ^r ), which is a resistance gene, into an appropriate exon in the same amino acid sequence frame. When this gene is introduced into mouse ES cells, it undergoes homologous recombination with the target gene region of mouse genomic DNA.
As shown in (A), the neo ^r gene is expressed by the promoter specific to the target gene and the poly A signal,
ES cells become resistant to G418 and proliferate. On the other hand, when the transgene is inserted at random locations of the genomic DNA, as shown in FIG. 9 (B), because it would be often no promoter nor a poly A signal in the vicinity, neo ^r gene also express Not, and thus such ES cells die by G418.

According to this method, it is possible to efficiently select homologous recombinant cells which can be obtained only at a frequency of 10 ^-3 from the gene-transfected cells, and it is actually possible to concentrate them about 10 times. ing. In addition, when a gene not originally expressed by ES cells is introduced, a promoter and poly A
We also proposed a method for constructing a transgene that incorporates another type of selectable marker gene (for example, the viral thymidine kinase gene) together with the signaled neo ^r gene, and obtaining homologous recombinant cells by two-step selection. (Eg, latest medicine, Vol. 46, No. 1, No. 12)
7-134, 1991).

[0011]

In the conventional target gene introduction method, a homologous recombinant can be obtained from a gene-introduced cell to some extent efficiently by performing selection using drug resistance as described above. However, as described above, in the method of incorporating a drug resistance gene or the like into a transgene to make a selection marker, the purpose of the target gene introduction method is homologous replacement of an arbitrary gene on an animal chromosome with a foreign gene. Considering this, it is not always preferable for the following two reasons.

The first reason is that in order to incorporate the drug resistance gene into the transgene, a part of the foreign gene to be introduced must be deleted. That is, FIG.
As shown in 0, in order to insert the neo ^r gene into the transgene and pair the other sequence region of the transgene with the homologous region of the ES cell genome to cause homologous recombination,
In order to ensure the homology with each other, a part of the foreign gene (the second exon in the case of FIG. 10) must be cut out and the neo ^r gene must be inserted therein.

The second reason is that since the homologous recombinant cells are selected using the expression of a drug resistance gene such as neo ^r as an index, the introduced drug resistance gene always remains in the recombinant ES cells. That's what it means. As described above, the conventional method cannot be said to be a complete gene replacement method in that the foreign gene cannot be introduced in the desired complete form and that the unwanted gene must be introduced. Met.

The present invention has been made in view of the above circumstances, and is a new gene replacement method capable of replacing a target gene with a gene having a desired structure and without leaving a selectable marker gene. Is intended to provide. Another object of the present invention is to provide a recombinant ES cell therefor.

[0015]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present invention provides at least one gene region of genomic DNA having resistance and sensitivity to each of two different antibiotics. Containing a foreign gene of
Provided is an embryonic stem cell characterized by being homologously recombined with an A sequence.

The present invention also provides that the target gene region of genomic DNA is homologously recombined with a DNA sequence containing at least one foreign gene that exhibits resistance and sensitivity to each of two different antibiotics. Existing embryonic stem cells,
Introduce a DNA fragment incorporating the gene of the desired structure,
DNA into which the homologous recombination sequence region of the above embryonic stem cell DNA has been introduced
A target gene replacement method characterized by introducing a gene of a desired structure into a target gene region of embryonic stem cell genomic DNA by homologous replacement with a fragment; and a target gene-substituted embryonic stem cell obtained by this method, And a transgenic animal derived from the embryonic stem cells.

The present invention further relates to an animal cell having a mutation sequence in any gene region of genomic DNA,
The mutant gene region of the animal cell is homologously replaced by a DNA sequence containing one or more foreign genes that are resistant and sensitive to each of the two different antibiotics to replace the normal gene of the mutant gene. By introducing the integrated DNA fragment into the above-mentioned replacement somatic cell and homologously replacing the homologous recombination sequence region of the replacement somatic cell DNA with the introduced DNA fragment, the mutant gene region of the animal cell genomic DNA becomes a normal gene. A target gene replacement method characterized by replacement is also provided.

According to the present invention, in the above-mentioned target gene replacement method, homologous recombinant cells are selected using the resistance to one antibiotic as an index, and homologous replacement cells are selected using the insensitivity to the other antibiotic as an index. This is also the preferred embodiment. Hereinafter, the present invention will be described in more detail. In the case of targeting ES cells, the target gene replacement method of the present invention first comprises ES as the first step.
Any gene region of the cell is homologously recombined with a DNA sequence containing one or more foreign genes that are resistant and sensitive to each of two different antibiotics.
At this stage, no structure intended for substitution is added to the DNA sequence. Recombinant cells can be selected as surviving cells against the antibiotic G418 in the same manner as in the conventional method, when the neo ^r gene, which is a drug resistance gene, is introduced into the DNA sequence. In this case, a drug susceptibility gene or a conditional lethal gene is inserted into the DNA sequence together with the drug resistance gene, but these genes are not used as selection markers for homologous recombinants at this stage.

FIG. 1 (A) is a schematic view illustrating a preferred embodiment of homologous recombination at the target gene recombination stage. Originally, ES cells sensitive to G418 is any genetic region of its genome DNA, neo ^r
When the gene is homologously recombined with a DNA sequence containing the herpes simplex virus thymidine kinase gene (hereinafter referred to as HSV-tk), the presence of the neo ^r gene changes its trait to G418-resistant cells. Therefore, homologous recombinant cells can be selected by recovering cells that survive in the presence of G418.

Then, as a second step, a gene DN in which a gene having a desired structure is inserted into the above homologous recombinant cell
The A fragment is introduced, and the above homologous recombination region on the ES cell genome is homologously recombined with the transgene DNA fragment. FIG. 1 (B) is a schematic view illustrating a preferred embodiment of such second-stage homologous recombination. In this case, the recombinant cells obtained by the homologous recombination in the first stage are
Since the HSV-tk gene is carried in the genomic DNA, when the antibiotic ganciclovir (GANC) is added to the culture solution, GANC is metabolized by the action of the HSV-tk gene, resulting in DNA elongation inhibition. Die. On the other hand, the cells homologously recombined with the transgene DNA fragment survive in the presence of GANC because the HSV-tk gene is no longer present in its genome.
Therefore, only homologous replacements with the transgene are selected. As shown in FIG. 1 (B), the neo ^r gene was completely eliminated from the ES cells by this second-stage homologous recombination.

As the second-stage selectable marker gene, in addition to a drug sensitivity gene such as HSV-tk, a conditional lethal gene such as FAS antigen gene can be used. In that case, an anti-FAS antibody can be used as an antibiotic having the same function as GANC for the HSV-tk gene, and a homologous recombinant can be obtained from cells insensitive to the anti-FAS antibody (surviving cells).

FIG. 2 is a schematic view illustrating another preferred embodiment of the target gene replacement method of the present invention. In this case, first, as shown in FIG. 2 (A), the guanine / xanthine / hypoxanthine / phosphoribosyl transferase (gpt) gene of Escherichia coli was ligated to the promoter (PGK) of the phosphoglycerol gene. Homologous recombination is carried out between the DNA sequence containing the and the target gene region of ES cells. Moreover, in this case, the hypoxanthine-phosphoribotuluyltransferase (h
prt) deficient strain is used.

That is, while cells of an organism synthesize their DNA via several pathways, hprt ^- cells do not function most of them, and only inosine 5'-
Monophosphate (IMP) → xanthosine 5′-monophosphate (X
MP) → guanosine 5′-monophosphate (GMP) → DNA
DNA is synthesized only by the above pathway. However,
Mycophenolic acid (MPA) is an IMP of the above pathway
→ Because hprt ⁻ cells block between XMP, M
In the presence of PA, DNA cannot be synthesized, and it will die. On the other hand, gpt catalyzes the synthesis of xanthine (X) into XMP, so that hprt ^- ES cells having this gpt gene are X → XMP → GMP → DN.
The A pathway functions and can survive in the presence of MPA. Therefore, in the method shown in FIG. 2 (A), the target gene region is PG
Cells can be selected which have been recombined with a DNA sequence containing K-gpt.

On the other hand, h having the gpt gene as described above
As described above, prt ⁻ cells have the function of the X → XMP function, and the cells are treated with the antibiotic 6-thioguanine (6-
When cultured in the presence of TG), the pathway of X → XMP is 6-T.
G is taken up and DNA synthesis is inhibited, resulting in death. Then, as shown in FIG. 2 (B), the region containing the gpt gene of the above recombinant cells is replaced with a transgene DNA fragment, and cells showing insensitivity to 6-TG are recovered. Somatic cells can be easily selected.

In the ES cells obtained by such two-step selection, the gene of interest (target gene) is completely replaced by the gene of the desired structure, and the gene used as a selection marker (for example, The neo ^r gene and the HSV-tk gene, or the gpt gene) also do not remain in the genome. Examples of the transgene include point mutation, deletion, antisense, and cD of the target gene.
It is possible to use all kinds of genes such as DNA sequences such as NA or the like, homologous genes of heterologous animals including humans and various mutant sequences thereof.

In this way, ES cells in which any gene is replaced with a gene of a desired structure can be injected into a blastocyst by ontogeny in a chimeric animal according to a conventional method, and further from its progeny. Transgenic animals can be obtained. Furthermore, the target gene replacement method of the present invention described above can be applied not only to ES cells but also to all tissue cells of mammals including humans. For example, if you take out cells of a tissue that loses normal function due to some gene deficiency, replace the defective gene with a normal gene and restore it to the original tissue, restore the normal function of that tissue. be able to. Such applications represent a new and effective means of gene therapy currently being carried out using, for example, retrovirus-infected cells. In addition, it can be applied to so-called missile therapy used for cancer treatment and the like.

The target gene replacement method of the present invention will be specifically described below with reference to examples. Needless to say, the present invention is not limited to the following examples.

[0028]

【Example】

Example 1 (Preparation of homologous recombinant ES cell) neo ^r gene and HS
The p53 gene of mouse ES cells (CCE or E14) was homologously recombined with the transgene DNA fragment incorporating the V-tk gene.

Undifferentiated cultured ES cells (8 × 10 ⁷ cells)
And transgene D by electroporation (about 500 μF)
The NA fragment was introduced at a ratio of 50 μg / 0.1 ml. As a result, about 30% of all ES cells survived, and gene transfer E
S cells were obtained. These ES cells were seeded on 30 plates, added with G418, and cultured for 7 days.
We obtained 54 colonies resistant to. These colonies were then individually separated and after culturing for a further 4 days, D
NA was extracted and homologous recombinant cells were confirmed by Southern blotting.

FIG. 3 is an example of the results, which is a Southern blot diagram (A) of the restriction enzyme HindIII fragment and p53.
It is a schematic diagram (B) which showed the structure of a gene. That is,
From the mouse p53 gene, two large and small HindIII fragments are blotted with the 3'probe at the position shown in FIG. 3 (B). Therefore, in the case of a non-homologous recombinant, two large and small clear bands are obtained as in clones 1, 2, 4, 6, 9 and the like in FIG. 3 (A). On the other hand, a smaller HindIII fragment is blotted with the 3'probe from the p53 gene which has undergone homologous recombination with a transgene having a HindIII recognition sequence at the 3'-terminal side in the sequence. And since such a homologous recombination gene exists in one of the diploids of the ES cell chromosome,
Like clones 3, 5, 7, 8, and 17 in FIG.
1/2 of two small fragments with slightly different molecular weights
You can get the amount.

4A and 4B similarly show p53.
The results obtained by blotting the EcoRI fragment of the gene with the 5'probe and the 3'probe, respectively, and the gene structure thereof. That is, since the p53 gene has EcoRI recognition sequences only at both ends, in the case of a non-homologous recombinant, it is detected as a single band by any probe. On the other hand, in the case of the homologous recombinant, since the EcoRI sequence is present in the transgene, the 5'probe and the 3'probe each yield two large and small bands in ½ amounts. However, as shown in FIG. 4 (A), in clones 32, 35 and 42, two bands were detected only by the 5'probe and only a single band was obtained by the 3'probe. Therefore, it was excluded from the homologous recombinants. Therefore, Southern blotting of this EcoRI fragment revealed clones 34, 36, 3
7, 43, 51, 53 and 54 were confirmed as homologous recombinants, and combined with the results shown in FIG. 3, a total of 12 clones of homologous recombinant cells were obtained. Example 2 (Preparation of Target Gene Substituted ES Cell) The recombinant gene region (p53 gene incorporating the neo ^r gene and the HSV-tk gene) of the homologous recombinant ES cell obtained in Example 1 was
It was replaced by a transgene DNA fragment incorporating the LacZ gene.

One of the 12 clones obtained in Example 1 ES cells: PFT201 (4 × 10 ⁷ cells) was electroporated (about 50 μF) to give 5 transgene DNA fragments.
It was introduced at a rate of 0 μg / 0.1 ml. As a result, about 3
0% survived, and transgenic ES cells were obtained. These cells were seeded on 30 plates, GANC was added and cultured for 7 days to obtain 48 colonies showing resistance to GANC. Then, these colonies were individually separated, and after further culturing for 4 days, DNA was extracted, and one clone of target gene-substituted somatic cell: PFZ252 was obtained by Southern blotting.

When the above procedure was repeated, another clone PFZ632 was obtained with good reproducibility. FIG. 5 (A) shows EcoRI obtained from the p53 gene region of ES cells, the homologous recombination region of PFT201, and the homologous substitution region of PFZ252 and PFZ632, respectively.
FIG. 5B is a Southern blot diagram of the fragment, and FIG. 5B is a schematic diagram showing the constitution of each gene region.

That is, P having an EcoRI recognition site
From FT201 and PFZ252 · 632, two large and small bands were obtained for the 5′-probe and the 3 ′ probe, respectively, but due to the difference in the EcoRI site shown in FIG. The band has a larger 5'fragment and a smaller 3'fragment than PFT201.

Further, as a result of another Southern blot analysis for PFZ252 and 632, these target gene regions were not detected when the neo ^r gene or HSV-tk gene was used as a probe, and only when the LacZ gene was used as a probe. It was detected as a clear band. The above results indicate that the p53 gene of ES cells was completely replaced by the transgene, and the neo ^r gene and HSV-t
This shows that the k gene has been deleted. Furthermore, these clones were cloned into X-gal as shown in FIG.
Since it was stained blue by staining, it was confirmed that the LacZ gene normally functions and expresses β-galactosidase. Example 3 (Creation of chimeric mouse) The target gene-substituted ES cell PFZ252 obtained in Example 2 was injected into the blastocyst of C57BL / 6J mouse by a conventional method, and transplanted into a foster mouse to develop into an individual. It was

As a result, 15 chimeric mice (♂11, ♀4) were obtained. Next, Southern blot analysis of the DNA extracted from the tails of these chimeric mice was performed to select individuals in which the p53 gene was replaced with the lacZ gene as desired, and this mouse was crossed with wild-type C57BL / 6J to obtain the primary trans. A transgenic mouse (F ₀ ) was obtained.

FIG. 7 (A) shows the above chimeric mouse and C57.
The result of Southern blot analysis of the DNA extracted from BL / 6J offspring (15) is shown. As is clear from FIG. 7, the DNA having two bands indicating that the lacZ gene region exists in one of the diploid chromosomes was
o. Obtained from 1-3, 6, 8, 11-14. Furthermore, individuals (♂, ♀) confirmed to be transgenic mice by the Southern blot analysis were selected from these F ₀ , and these were crossed to obtain a second-generation transgenic mouse (F ₁ ). .

FIG. 8 shows the child (15) of the above F ₀ (♂, ♀).
The result of Southern blot analysis of the DNA extracted from As is clear from FIG. 8, lacZ was found in the descendants (F ₁ ) of the transgenic mouse (F ₀ ) and the fellows.
Individuals without genes (Nos. 1, 3, 6, 7, 1
0, 11, 14), an individual having the lacZ gene on one of the chromosomes (Nos. 2, 8, 9, 12, 15), and a homozygous individual having the lacZ gene on both the chromosomes (No.
s. 4, 5) were obtained.

From the above results, mouse ES cells in which the p53 gene region was homologously replaced by the lacZ gene were obtained by the method of the present invention, and the lacZ gene was stably integrated into at least one of the chromosomes from the ES cells. It was confirmed that transgenic mice were obtained.

[0040]

As described in detail above, the target gene replacement method of the present invention makes it possible to reliably and efficiently replace an arbitrary gene of an ES cell with a gene having a desired structure. Substitute E obtained by this method
Transgenic animals derived from S cells make it possible to analyze gene expression of any kind at the individual level. In addition, by applying the method of the present invention to somatic cells of humans and the like, safe and effective gene therapy becomes possible.

[Brief description of drawings]

1 (A) and (B) are schematic diagrams showing examples of target gene homologous recombination in the method of the present invention and replacement thereof, respectively.

2 (A) and (B) are schematic diagrams showing another example of target gene homologous recombination and the replacement thereof in the method of the present invention, respectively.

FIG. 3 is a Southern blot diagram (A) against a HindIII fragment of the homologous recombination region and a schematic diagram showing the structure of this gene region.

FIG. 4 is a Southern blot diagram (A) against an EcoRI fragment of the homologous recombination region and a structural schematic diagram of the gene region.

FIG. 5 is a Southern blot diagram (A) of EcoRI fragments in the homologous recombination region and the homologous substitution region, and a schematic diagram showing the structure of the gene region.

FIG. 6 is a micrograph showing X-gal staining of ES cells in which p53 gene was replaced by lacZ gene.

7 (A) and (B) are Southern blot diagrams of DNA extracted from the transgenic mouse obtained by the present invention, respectively.

FIG. 8 is a schematic diagram showing an example of producing a transgenic mouse by the target gene introduction method.

FIG. 9 is a schematic view illustrating a method for selecting a homologous recombinant in the conventional target gene introduction method.

FIG. 10 is a schematic diagram illustrating the structure of transgene in a conventional target gene transfection method and its homologous recombination process in ES cells.

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Claims (22)

[Claims] 1. An arbitrary gene region of genomic DNA is 2
An embryonic stem cell characterized by being homologously recombined with a DNA sequence containing one or more foreign genes that are resistant and sensitive to different types of antibiotics. 2. The embryonic stem cell according to claim 1, wherein the foreign gene in the DNA sequence is a drug resistance gene, a drug sensitivity gene and / or a conditionally lethal gene. 3. The embryonic stem cell according to claim 2, wherein the foreign drug resistance gene is a Neo resistance gene. 4. The embryonic stem cell according to claim 2, wherein the foreign drug susceptibility gene is a herpes simplex virus thymidine kinase gene. 5. The embryonic stem cell according to claim 2, wherein the exogenous conditionally lethal gene is the FAS antigen gene. 6. The embryonic stem cell according to claim 1, wherein the embryonic stem cell is a hypoxanthine phosphoribosyltransferase-deficient strain, and the foreign gene in the DNA sequence is a guanine xanthine hypoxanthine phosphoribosyltransferase gene of Escherichia coli. . 7. An embryonic stem cell in which a target gene region of genomic DNA is homologously recombined with a DNA sequence containing at least one foreign gene that is resistant and sensitive to each of two different antibiotics. The above-mentioned embryonic stem cell DN is introduced by introducing a DNA fragment into which a gene having a desired structure is incorporated.
A target gene replacement method, which comprises replacing the target gene region of embryonic stem cell genomic DNA with a gene having a desired structure by homologously replacing the homologous recombination sequence region of A with the introduced DNA fragment. 8. A homologous recombinant cell is selected using resistance to one antibiotic as an index, and a homologous replacement cell is selected using insensitivity to the other antibiotic as an index.
Target gene replacement method. 9. The target gene replacement method according to claim 7, wherein the foreign genes in the DNA sequence are a drug resistance gene, a drug sensitivity gene and / or a conditionally lethal gene. 10. The target gene replacement method according to claim 9, wherein the foreign drug resistance gene is a Neo resistance gene. 11. The method for target gene replacement according to claim 9, wherein the foreign drug susceptibility gene is the thymidine kinase gene of herpes simplex virus. 12. The target gene replacement method according to claim 9, wherein the exogenous conditionally lethal gene is the FAS antigen gene. 13. The embryonic stem cell is a hypoxanthine / phosphoribosyltransferase-deficient strain, and the foreign gene in the DNA sequence is a guanine / xanthine / hypoxanthine / phosphoribosyltransferase gene of Escherichia coli.
Or 8 target gene replacement methods. 14. A target gene-substituted embryonic stem cell obtained by the method according to claim 7. 15. A transgenic animal derived from the target gene-replaced embryonic stem cell according to claim 14. 16. An animal cell having a mutant sequence in an arbitrary gene region of genomic DNA, wherein the mutant gene region of the animal cell exhibits resistance and sensitivity to each of two different antibiotics. A DNA fragment that has undergone homologous recombination with the above-mentioned DNA sequence containing a foreign gene, and has introduced a normal gene of a mutant gene into the recombinant cell, and has introduced a homologous recombination sequence region of the recombinant cell DNA. A target gene replacement method, which comprises replacing a mutant gene region of an animal cell genomic DNA with a normal gene by homologously replacing with. 17. The target gene replacement method according to claim 16, wherein homologous recombinant cells are selected using the resistance to one antibiotic as an index, and homologous replacement cells are selected using the insensitivity to the other antibiotic as an index. 18. The target gene replacement method according to claim 16 or 17, wherein the foreign genes in the DNA sequence are a drug resistance gene, a drug sensitivity gene and / or a conditionally lethal gene. 19. The target gene replacement method according to claim 18, wherein the foreign drug resistance gene is a Neo resistance gene. 20. The foreign drug susceptibility gene is a herpes simplex virus thymidine kinase gene.
Target gene replacement method. 21. The target gene replacement method according to claim 18, wherein the exogenous conditionally lethal gene is the FAS antigen gene. 22. The embryonic stem cell is a hypoxanthine / phosphoribosyltransferase-deficient strain, and the foreign gene in the DNA sequence is a guanine / xanthine / hypoxanthine / phosphoribosyltransferase gene of Escherichia coli.
6 or 17 target gene replacement methods.